Functional Conservation of a Root Hair Cell-Specific cis-Element in Angiosperms with Different ...

14 Pages·2006·1.82 MB·English

ThePlantCell,Vol.18,2958–2970,November2006,www.plantcell.orgª2006AmericanSocietyofPlantBiologists
Functional Conservation of a Root Hair Cell-Specific
cis-Element in Angiosperms with Different Root Hair
Distribution Patterns W
DongWookKim,aSangHoLee,aSang-BongChoi,bSu-KyungWon,aYoon-KyungHeo,aMisukCho,aYoun-IlPark,a
andHyung-TaegChoa,c,1
aDepartmentofBiology,ChungnamNationalUniversity,Daejeon305-764,Korea
bDepartmentofBiologicalSciences,MyongjiUniversity,Yongin,Kyunggido449-728,Korea
cEnvironmentalBiotechnologyNationalCoreResearchCenter,GyeongsangNationalUniversity,Jinju660-701,Korea
Vascularplantsdevelopdistinctiveroothairdistributionpatternsintherootepidermis,dependingonthetaxon.Thethree
patterns, random (Type 1), asymmetrical cell division (Type 2), and positionally cued (Type 3), are controlled by different
upstreamfate-determiningfactorsthatmediateexpressionofroothaircell-specificgenesforhairmorphogenesis.Here,we
addresswhethertheseroothairgenespossessacommontranscriptionalregulatorymodule(cis-element)determiningcell-
typespecificitydespitedifferencesinthefinalroothairpattern.WeidentifiedArabidopsisthalianaexpansinA7(AtEXPA7)
orthologous(andparalogous)genesfromdiverseangiospermspecieswithdifferenthairdistributionpatterns.Thepromoters
of these genes contain conserved root hair–specific cis-elements (RHEs) that were functionally verified in the Type-3
Arabidopsisroot.ThepromoterofAtEXPA7(Type-3pattern)alsoshowedhaircell–specificexpressionintheType2rice(Oryza
sativa)root.Roothair–specificgenesotherthanEXPAsalsocarryfunctionallyhomologousRHEsintheirpromoters.TheRHE
coreconsensuswasestablishedbyamultiplealignmentoffunctionallycharacterizedRHEsfromdifferentspeciesandbyhigh-
resolutionanalysisofAtEXPA7RHE1.Ourresultssuggestthatthisregulatorymoduleofroothair–specificgeneshasbeen
conservedacrossangiospermsdespitethedivergenceofupstreamfate-determiningmachinery.
INTRODUCTION cells,longandshort(alongtherootlongitudinalaxis),andonlythe
shortcellsdifferentiateintohaircells.Type2patterningoccursin
Roothairsaretubularoutgrowthsofcertainrootepidermalcells.
lower ferns, monocots, and basal angiosperm families (Figure
Because they constitute the majority of the root surface area,
1B). In addition to these basic root hair patterns, root hairs
roothairsarethoughttocontributetotheprimaryrootfunctions
originatingfromthecortex(Pinkerton,1936)andaType3variant,
ofabsorption,anchorage,andsoilmicrobeinteractions(Grierson
inwhichlonghairsarisefromtheHpositionandshorthairsfrom
andSchiefelbein,2002).Thefatedeterminationanddistribution
theNposition(Tsaietal.,2003),havebeendescribed.
patternofhairandnon-haircellshasbeenintensivelystudiedin
Differencesinhaircelldistributionpatternsindicatethatdifferent
Arabidopsis. Root hair cells are located over two underlying
cellfatedeterminingsystemsoperateinthedifferentpatterntypes.
cortical cells (the H cell position), whereas non-hair cells are
InArabidopsisthaliana,whenahaircellwaslaserablatedandthe
positionedoverasinglecorticalcell(theNcellposition)(Dolan
Hpositionwasthenoccupiedbyaneighboringnon-haircell,the
etal.,1993;Galwayetal.,1994).Thisposition-dependenthaircell
non-haircellcould differentiate a roothair(Berger etal.,1998),
specification resultsin rowsofhair cellsalong thelongitudinal
demonstrating that a positional cue determines the Type 3 cell
rootaxisandhasbeenfoundinBrassicaceaeandothereudicot
fate.ThetranscriptionalregulatorynetworkunderpinningType3
families(Cormack,1947;Clowes,2000;DolanandCosta,2001;
fatedeterminationhasbeencharacterizedinArabidopsis.Briefly,
Figure1).Thisstripedpattern(Type3)isoneofthreetypesofhair
theexpressionofGLABRA2(GL2;ahomeodomaintranscription
cell distribution patterns (Leavitt, 1904; Cormack, 1937, 1947;
factor)ismodulatedbytheMYB/bHLH/WD40complex(forrecent
CutterandFeldmann,1970;CutterandHung,1972;Dolan,1996;
review,seeSchiefelbeinandLee,2006)orbychromatinremod-
Clowes,2000;Dolan andCosta, 2001;Figure1A).IntheType
eling (Costa and Shaw, 2006) in a position-dependent manner.
1pattern,roothaircellscandifferentiatefromanyepidermalcell.
GL2 suppresses genes required for hair differentiation in the N
Mostdicots,manymonocots,andmostfernsshowthispattern.
position(Ohashietal.,2003).Incontrastwithposition-dependent
IntheType2pattern,therootepidermisconsistsoftwosizesof
Type3patterning,Type2patternsdependonasymmetricalcell
division. Hair-forming cells (trichoblasts) in Type 2 roots derive
from a late, unequal transverse cell division in the epidermal
1To whom correspondence should be addressed. E-mail htcho@cnu.
ac.kr;fax[phone]. stemcell(Cormack,1944;Clowes,2000).Theshorterofthetwo
The author responsible for distribution of materials integral to the daughtercellsbecomesatrichoblast,whichisnoticeablyrichin
findingspresentedinthisarticleinaccordancewiththepolicydescribed cytoplasmanddeeplystainedbydyes.Unequalcelldivisionmay
intheInstructionsforAuthors(www.plantcell.org)is:Hyung-TaegCho
asymmetricallypartitioncellfatedeterminantsasobservedinsuch
([email protected]).
WOnlineversioncontainsWeb-onlydata. diverseorganismsasbacteria,yeast,worm,insects,andplants
www.plantcell.org/cgi/doi/10.1105/tpc.106.045229 (Scheres and Benfey, 1999; Betschinger and Knoblich, 2004;
ConservationofaRootHaircis-Element 2959
and Tierney, 2000), At LRX1 and At LRX2 (encoding Leu-rich
extensins;Baumbergeretal.,2001,2003),andAtEXPA7andAt
EXPA18(encodingexpansins;ChoandCosgrove,2002).These
genes show almost identical spatio-temporal expression pat-
ternsindifferentiatingroothaircellsandarethoughttoinduce
alterations in cell wall assembly during hair morphogenesis.
These downstream genes are controlled byupstream fate de-
terminantsandothercuesintheType3patterninArabidopsis
(Bernhardt andTierney,2000;ChoandCosgrove,2002).Root
hairsoccurinallgroupsofvascularplants,andcommoncellular
and molecular behaviors are observed across different species
duringhairdevelopment(PetersonandFarquhar,1996;Ridgeand
Emons, 2000), implying that root hair–specific genes are func-
tionallyconservedinplants.Here,weaskedwhetherroothaircell-
specificgenesindifferenthairdistributiontypesaremodulatedby
independent orconvergentregulatorypathways(that is, bydif-
ferentorcommoncis-andtrans-elements).Toaddressthisissue,
wehavespecificallyinvestigatedthefollowingquestions:(1)Are
orthologs of root hair–specific genes present in species having
differenthairdistributiontypes?(2)Arethoseorthologousgenes
alsoroothairspecific?(3)Iftheyareroothairspecific,dotheir
promotersincludeacommonoratype-specificcis-elementfor
roothairspecificity?(4)Ifthosegenesuseacommoncis-element
forroothairspecificity,isthiscis-elementfunctionallycompatible
betweenroothairpatterningtypes?
Inaddition,thisstudywasalsodesignedtodemonstrateex-
perimentallytheconservationofacelltype–specificcis-element
over angiosperm evolution. A plethora of genomic data have
enabled computational predictions of conserved regulatory
noncoding elements from orthologous and paralogous genes
(Gumucioetal.,1992;Jareborgetal.,1999;Wassermanetal.,
2000;Kaplinskyetal.,2002;GuoandMoose,2003;Hongetal.,
2003;Inadaetal.,2003;Bermanetal.,2004;Habereretal.,2004).
However,thecomputationalmethodcanbeeffectiveonlywhen
combined with experimental analysis, which has rarely been
conductedinplants(Kellogg,2004).
In this study, we have identified genes orthologous and
paralogoustoAtEXPA7fromangiospermspecieswithdifferent
hairdistributionpatterns,functionallydefinedaroothair–specific
cis-element,and demonstratedthe abilityofthiscommoncis-
element to drive root hair cell-specific expression of genes in
rootswithdifferentdistributionpatterns.Theresultsindicatethat
Figure 1. Three Root Hair Patterning Types and Their Phylogenetic thegeneregulatorymodule(cis-andtrans-elements)forroothair
RelationshipinTracheophytes. specificity has been conserved despite the divergence of up-
(A)Schematicdiagramofthethreeroothairpatterningtypes.Densely streamfate-determiningmachineryinangiosperms.
stainabletrichoblastcellsarecoloredinType2andType3.Smallcircles
indicateroothairs.
(B)Thephylogeneticrelationshipofroothairdistributiontypes.Thetaxon RESULTS
(order)includingType2isinredandType3inblue.Thephylogenetictree
oftracheophyteswasmodifiedfromJuddetal.(1999).Roothairdistri-
High-ResolutioninVivoAnalysesoftheRootHair–Specific
butiontypesofthetaxawerereferredtothesurveybyClowes(2000).
cis-ElementinRepresentativePromoters
Previouslydefinedpromoterregionsconferringroothairspec-
Haecker et al., 2004). The Type 1 pattern may be either an ificity in At EXPA7 and At EXPA18 (Cho and Cosgrove, 2002)
ancestralpatternoradegeneratepatternderivedfromType2or were further dissected to determine the structure of the core
Type3(DolanandCosta,2001). cis-element(RHEforroothairelement).Thepresenceofaddi-
Several root hair cell-specific genes have been identified in tional enhancing elements and the function of multiple RHEs
Arabidopsis: AtPRP3 (encoding a Pro-rich protein; Bernhardt were investigated inthepromoters ofthe twoparalogous root
2960 ThePlantCell
Figure2. AnalysesofP ,P ,andTheirRootHairCell-Specificcis-Elements(RHEs)inArabidopsis.
AtEXPA7 AtEXPA18
(A)NucleotidesequenceoftheP RHE1(E1inFigure5)regionandsubstitutednucleotides(lowercase)in4-and1-bpmutations.Numbersabove
AtEXPA7
thewild-typesequenceindicaterelativedistancefromthetranscriptioninitiationsite.TheTATAboxisshowninbold.
(B)A10-bpdeletionanalysisoftheproximalupstreamregionofRHE1.Inset:fluorescenceimageofthe-134:GFProot.
(C)GOFanalysisofRHEsofP .FluorescenceimagesofthisGOFanalysisareshowninSupplementalFigure1online.
AtEXPA7
ConservationofaRootHaircis-Element 2961
hair–specificexpansingenes.Finebase-substitutionanalysesof
thepromotersofAtEXPA7(P )andAtEXPA18(P )
AtEXPA7 AtEXPA18
at 1- to 10-bp resolution (Figures 2A and 2F) revealed that a
similar sequence motif was essential for RHE function in both
promoters,regionsM1toM5inP andE18M5toE18M7in
AtEXPA7
P (Figures2D,2E,and2G).A4-bpsubstitutionanalysis
AtEXPA18
oftheP RHE1regionsharplydividedthesequenceinto
AtEXPA7
twofunctionalpartscenteringaroundM3;mutationofM3didnot
significantlyaffecttheRHEfunction(Figure2D).A1-bpsubsti-
tutionanalysisidentifiedindividualnucleotidesrequiredforthe
function of P RHE1 (Figure2E) and revealed the strictly
AtEXPA7
conservedCACGmotifintheRHE.MultipleRHEssharingthis
common sequence function additively in promoter activity in
bothgenes(Figures2Cand2H).Regions-110/-90and-241/-196
(relativetothetranscriptionstartsite)ofP andP ,
AtEXPA7 AtEXPA18
respectively, may include some elements that enhance RHE
activity(Figures2B,2G,and2H).Thefulldescriptionofpromoter
and RHE analyses of At EXPA7 and At EXPA18 is provided in
SupplementalText1online.
EXPA7OrthologsfromDiverseAngiospermswith
DifferentRootHairPatterns
OurnextquestionwaswhethergenesorthologoustoAtEXPA7
(orAtEXPA18)existinspecieshavingdifferentroothairdistri-
bution types. A BLAST search using At EXPA7 to query the
databases identified closely related sequences from cabbage
(Brassicaoleracea)(Type3;BoEXPA7),rice(Oryzasativa)(Type2;
OsEXPA17andOsEXPA30),andMedicagotruncatula(Type1;
MtEXPA7andMtEXPA8).Theroothairtypesweredetermined
by direct observation. EXPA7 orthologs from other species
wereobtainedbyPCRusingprimersfromconservedregionsof
theabovesequences(seeSupplementalTable1online).Coding
sequenceswereclonedfrombarley(Hordeumvulgare)(Type1;
HvEXPA7),wheat(Triticumaestivum)(Type2;TaEXPA7),maize Figure3. Neighbor-JoiningPhylogeneticTreeofExpansinProteinSe-
(Zeamays)(Type1;ZmEXPA7),radish(Raphanussativus)(Type3; quences.
RsEXPA7),celandine(Chelidoniummajus)(Type1;CmEXPA7), Theunrootedtree,constructedusingMEGA3.0,demonstratesthetight
andbalsam(Impatiensbalsamina)(Type3;IbEXPA7).Theroot evolutionaryrelationshipofEXPA7orthologsintheexpansinfamily(shaded
hairtypesareasdescribedbyClowes(2000). ingray).Thetreeincludes75EXPAsfromdiverseplanttaxaandAtEXPB2
Theaminoacidsequencebetween55(Gly)and181(Ile)ofAt asanoutgroupandwasconstructedafterCLUSTALWalignmentofthe
EXPA7 and the equivalent regions of each expansin protein aminoacidsequencebetween55(Gly)and181(Ile)ofAtEXPA7andthe
equivalentregionsofeachprotein.Bootstrapvalues(1000replicates)for
sequence were aligned by CLUSTAL W. A neighbor-joining
thistreeareshowninSupplementalFigure3online.Multiplealignmentsfor
phylogenetictreewasgeneratedfromthisalignment(Figure3).
thistreeareshowninSupplementalFigure4online.At,Arabidopsisthaliana;
The tree shows that these EXPA7-related expansins (EXPA7s)
Bo,Brassicaoleracea;Cm,Chelidoniummajus;Hv,Hordeumvulgare;Ib,
are a monophyletic group. Of 1000 bootstrap replicates, 67%
Impatiensbalsamina;Mp,Marchantiapolymorpha;Mq,Marsileaquadrifolia;
supporttheestablishmentoftheEXPA7clade(seeSupplemental Mt,Medicagotruncatula;Os,Oryzasativa;Pp,Physcomitrellapatens;Pt,
Figure3online).GraminaceaeEXPA7s,exceptforOsEXPA17, Pinustaeda;Rd,Regnellidiumdiphyllum;Rs,Raphanussativus;Ta,Triticum
form a tight subclade distinct from the eudicot EXPA7s. Os aestivum;Zm,Zeamays.Bar¼substitutions/site.
Figure2. (continued).
(D)A4-bpsubstitutionanalysis(M1toM8asshownin[A])oftheRHE1region.
(E)A1-bpsubstitutionanalysis(1to16asshownin[A])oftheRHE1core.
(F)NucleotidesequenceoftheP RHE1(E1inFigure5)regionandsubstitutednucleotides(lowercase)inpromotermutations(E18M1toE18M8).
AtEXPA18
Numbersabovethewild-typesequenceindicatetherelativedistancefromthetranscriptioninitiationsite.
(G)SubstitutionanalysisoftheregionsurroundingRHE1.
(H)GOFanalysisofRHE1.FluorescenceimagesofthisGOFanalysisareshowninSupplementalFigure2online.
‘‘Cont’’in(B)to(E),(G),and(H)showsthepromoteractivityofPm35S:GFPtransformants.ErrorbarsindicateSE;numberoftransgenicsisindicatedinMethods.
Figure4. RHEsAreFunctionallyConservedinEXPA7OrthologsfromSpecieswithDifferentRootHairPatternTypesandinArabidopsisRootHair
GenesOtherThanExpansins.
Deleted(-),substituted(-mE),orGOF((cid:2)/(cid:2))promoterfragmentsofEXPA7orthologsfromrice([A]and[B]),wheat(C),maize(D),M.truncatula(E),
balsam(F),andcabbage(G)werefusedtoGFPandanalyzedinType3Arabidopsisroots.ThepromoterfragmentsofotherArabidopsisroothairgenes
(AtLRX1[H]andAtPRP3[I])alsowerefusedtoGFPandanalyzedinArabidopsis.InsetsarefluorescenceimagesoftheArabidopsisrootcontainingthe
designatedpromoterconstructs.ErrorbarsindicateSE;numberoftransgenicsisindicatedinMethods.
ConservationofaRootHaircis-Element 2963
(http://meme.sdsc.edu/meme/meme.html) was used to lo-
cate putative RHE regions in the promoters. Deletion, base
substitution, and gain-of-function (GOF) constructs of the
orthologouspromotersfusedtothegreenfluorescentprotein
(GFP)genewere introducedintoArabidopsisplants(Type3)
byAgrobacteriumtumefaciens–mediatedtransformation,and
root hair–specific promoter activity was analyzed in vivo
(Figure4).
AlltheEXPA7orthologouspromotersincludedmultipleRHEs
intheproximalpromoterregionandweresimilartoRHEsfrom
P and P , including the hallmark CACG motif
AtEXPA7 AtEXPA18
(Figure 5). Despite the different root hair types from which the
promoters originated, the orthologous promoters showed root
hair cell-specific expression of GFP in the Type 3 Arabidopsis
root (Figures 4A to 4G). Deletion or base substitution of RHEs
greatly decreased the activity of the orthologous promoters.
GOF analyses of representative orthologous promoters dem-
onstrated that the RHE fragments were sufficient to drive root
hair–specific gene expression (Figures 4A, 4E, and 4F; see
SupplementalFigure5online).Theseresultscollectivelysuggest
that RHE-mediated regulation of root hair–specific genes has
been conserved, even though the upstream fate-determining
componentshavediverged.
Figure5. RHEsfromEXPA7OrthologsandSeveralRootHair–Specific
Genes.
Numbersaboveeachsequenceindicatethedeletionconstructsusedfor
promoteranalyses.Numbersbelowthesequencearestartingnucleotide
positionsoftheRHEcore(blackbox),andthelargernumbersabovethe
RHEcoreindicatethespacerlength(twoorthreenucleotides).Thegray
boxesindicatetheconservedRHEextensionsinGraminaceaeP s.
EXPA7
Nucleotidepositionsarenumberedrelativetotheputativetranscription
initiationsiteorstartcodon(ATG).Theroothaircelldistributiontypeis Figure 6. The RHE-Containing Promoters from Type 2 and Type 3
designatedinparenthesesafterthegenename.An‘‘r’’aftertheRHE PatternGenesAreAlsoRootHairSpecificintheType2RiceRoot.
numberindicatesareverseorientationoftheRHE,andaquestionmark
Confocal microscopy images of transgenic rice roots harboring
indicatesthattheRHEhasnotbeenexperimentallyverified.
P (-318):GFP([A]to[C])andP (-386):GFP(D).
OsEXPA30 AtEXPA7
(A)Greenfluorescenceisshownindifferentiatinghaircells(trichoblasts).
EXPA17islocatedatthebaseoftheEXPA7clade,indicatingits Redfluorescenceshowsthepropidiumiodide–stainedcellwall.
earlydivergencefromtheclade. (B)Greenfluorescencefrommatureroothairs.
(C)Reconstitutedconfocalcross-sectionalviewofthericerootexpress-
ingP :GFP.TheGFP-expressinghaircellsarecontactingeithera
OsEXPA30
RHEsofEXPA7OrthologsAreFunctionallyConserved singleortwounderlyingcorticalcells(asterisks).
(D)ArabidopsisEXPA7promoterisalsoactiveinthericeroothaircells
Promoter sequencesof EXPA7orthologs wereobtained either butfollowstheType2pattern.Greenfluorescenceisfoundonlyinthe
fromdatabasesorbyPCR-basedcloning.TheMEMEalgorithm roothaircells.Bars¼20mm.
2964 ThePlantCell
RHEsofOtherArabidopsisRootHairGenesAre root hairs (Baumberger et al., 2001). The P (-134):LRX1
AtEXPA7
FunctionallyConserved constructwasintroducedintolrx1mutantplants.TheP -
AtEXPA7
drivenexpressionofwild-typeAtLRX1successfullyrescuedthe
Threeadditionalroothair–specificgeneshavebeenreportedin
roothairdefectsinlrx1plants(Figure7).Thisresultsuggeststhat
Arabidopsis, At LRX1, AtLRX2 , and At PRP3 (Bernhardt and
RHEs are functionally compatible between different root hair
Tierney,2000;Baumbergeretal.,2001,2003).Thesegenesare
genes.
expressedonlyinroothaircells;genesthatareexpressedbothin
haircellsandothercelltypesoftherootarenotincludedinthis
designation. These root hair–specific genes show the same ConstructionoftheRHEConsensus
expressionpatternasAtEXPA7andAtEXPA18,andwetherefore
Theconsensus RHEwasestablished bothbyamultiple align-
analyzedtheroothair–specificcis-elementofthesegenes.Pro-
mentofRHEsfromEXPA7genesandotherroothairgenesand
motersoftheseadditionalroothairgenescontainedRHE(s)inthe
byfunctionalanalysesofP RHE1(AtEXPA7E1).TheRHE
proximal promoter region: two reverse RHEs in P , one AtEXPA7
AtLRX1
coreconsistsof16to17bp,whichisdividedintotwoconserved
reverseRHEinP ,andonereverseRHEinP (Figure5).
AtLRX2 AtPRP3
partsjoinedbyashortlinkerregionthatincludesoneadditional
DeletionandbasesubstitutionanalysesofRHEsinP and
AtLRX1
nucleotideinsomeRHEs.Conservationofthe16-or17-bpRHE
P demonstratedthattheRHEmotifisrequiredfortheroot
AtPRP3
core,relativetoflankingregions,issuchthatfewerthanthreeof
hairspecificityofthesepromoters(Figures4Hand4I).Thesedata
thefournucleotidespeciesorpredominantnucleotidespecies
indicatethatotherroothair–specificgenesinArabidopsisexploit
occurinagivenposition,withtheexceptionofthelinkerregion
thesamecis-elementtodrivecelltype–specificexpression.
(Figure8A).ThetwoconservedpartsoftheRHEcoreappearto
AfulldescriptionofthepromoteranalysesofEXPA7orthologs
form an incomplete palindrome of each other (Figure 8C). The
and other Arabidopsis root hair genes is presented in Supple-
sequenceconservationisstrongerintherightpart(RP)thanin
mentalText2online.
theleftpart(LP).RPistypicallyrepresentedbythesignatureG(/
T)CACGT(/A),inwhichtheCACGmotifisstrictlyconservedinall
P andP AreAlsoRootHairSpecificinthe
OsEXPA30 AtEXPA7 the functionally verified RHEs (Figure 8A). Any single base
Type2RiceRoot
substitutioninthisCACGmotifabrogatedthepromoteractivity
To determine whether the rice EXPA7 ortholog promoter is almostcompletely(Figure2E).InLP,onlytheTGmotifishighly
operative in rice root hair cells and whether the Arabidopsis conserved,andsomepartialconservationsarefoundindifferent
(Type3)EXPA7promoterretainsroothaircellspecificityinType RHEsubgroups.TintheTGmotifisconservedinallRHEs,and
2riceroots,P (-318):GFPandP (-386):GFPwere its replacement was fatal to promoter activity (Figure 2E). Al-
OsEXPA30 AtEXPA7
introduced into rice embryonic calli using Agrobacterium- though the G of this TG is conserved in most RHEs, it can be
mediatedtransformation,andthetransgenicseedlingrootswere functionallyreplacedwithAorC,asshowninAtEXPA7E3and
examined (Figure 6). P -driven GFP expression was MtEXPA7E3r,respectively(Figure8A).Thepromoterswiththese
OsEXPA30
detectablespecificallyinthehaircellsandinafewcellsbefore minorvariantRHEswerestillroothairspecific(Figures2C,-386/-
the hair bulge started to emerge (Figure 6A). The expression 200,and4E,-78;seeSupplementalFigure1online,-386/-200).A
pattern was consistent with the Type 2 hair cell distribution, pointmutationatthisposition(no.8in1-bpmutations;Figure2A)
wherelongandshortepidermalcellsalternatealongthelongaxis inAtEXPA7E1affectedthepromoteractivitymuchlessthandid
oftheroot,andtheshortercellsbecomehaircells.Moreover,the amutationintheTposition(no.7)(Figure2E).
rice Type 2 hair cells were located on either a single or two The linker between LP and RP varies in length, containing
underlying cortical cells (Figure 6C), clearly distinct from the eithertwoorthreenucleotides,andwerefertotheselinkersas
position-dependentType3pattern.ThisType2patterninrice two-nucleotideorthree-nucleotidespacers(Figure8C).Totest
appears to be slightly disturbed in the upper hair maturation theinfluenceofthespacersonRHEfunction,wemadea1-bp
zone,whereconsecutivehaircellswereoccasionallyobserved deletionfromthethree-nucleotidespacerinAtEXPA7E1anda
(ourunpublisheddata).TheP activitywaslimitedtoroot
OsEXPA30
hair cells, at least in seedlings. This result indicates that Os
EXPA30 is a functional equivalent to At EXPA7 in rice. The At
EXPA7 promoter also wasroothair cellspecific in rice (Figure
6D).Theresultsfromtransgenicriceplantssuggestthatthecis-
element for root hair specificity is compatible between Type 2
and Type 3 species despite differences in upstream fate-
determiningmechanismsbetweenthetwotypes.
Thelrx1MutantCouldBeRescuedbyP -Driven
AtEXPA7
Figure7. TheAtEXPA7PromoterCanFunctionallySubstitutefortheAt
ExpressionofAtLRX1
LRX1Promoter.
We tested whether the PAtEXPA7 promoter is functionally com- (A)AberrantroothairphenotypeoftheArabidopsislrx1mutant.
patiblewiththepromotersofotherroothairgenes.TheArabi- (B)IntroductionofP (-134):LRX1intothelrx1mutantrescuesthe
AtEXPA7
dopsislrx1mutanthasfrequentlyaborted,swollen,orbranched roothairphenotype.Bar¼100mmfor(A)and(B).
ConservationofaRootHaircis-Element 2965
Figure8. TheRHEConsensus.
(A)AlignmentofRHEsequences.Conservednucleotidesareshaded.Numbersabovethealignmentindicatethenucleotidepositionintheconsensus.
(B)SequencelogooftheRHEregionillustratingconservednucleotidesintallerletters.TheRHEconsensusisunderlined.Thenucleotidesequencelogo
wascreatedbytheWebLogoprogram(Crooksetal.,2004;http://weblogo.berkeley.edu/).
(C)StructureoftheRHEconsensus.Twoinvertedarrowsindicatetheimperfectpalindromicsequences.Strictlyconservednucleotidesareinbold.The
‘‘n’’indicatesanynucleotide.
1-bp insertion into the two-nucleotide spacer in AtEXPA18E1. P , the same direction for multiple RHEsin a given pro-
MtEXPA7
The1-bpinsertionordeletionwasplacedinthenonconserved moterisnoticeablypreferred.
RHE linker region of the -241 and -134 deletion promoters of
P andP ,respectively(Figures9Aand9B).Abase
AtEXPA18 AtEXPA7
DISCUSSION
pair addition in the At EXPA18E1 linker greatly decreased the
promoteractivity,anda1-bpdeletionintheAtEXPA7E1linker
Studiesofgeneregulationatthetranscriptionallevelrequirethe
caused a decrease of ;60% in promoter activity. These data
characterizationofcis-regulatoryelements,trans-actingfactors,
indicate that the linker length is critical to RHE function, even
andtheirinteractionsinregulatingtranscription.Theprerequisite
thoughnucleotidesubstitutionofthelinkerisnot.
to such studies isthe identification of functional cis-regulatory
sequencesthatarelocatedinnoncodingDNAregionsatvarying
distances from the transcription start site. A cis-element pro-
OrientationofRHE
vides the ultimate information controlling where, when, and at
Inmanyenhancerelements,aforwardorreverseorientationis whatlevelageneisexpressed.Althoughtherearemanycom-
equallyfunctional(Lewin,2004).Twelveof29putativeorfunc- putational predictions and a few experimental tests of whole
tionally identified RHEs are in the reverse direction (Figure 5). promoters, we currently have a paucity of examples of well-
AlthoughbothforwardandreverseRHEsarefoundtogetherin characterized cis-regulatory elements from either plants or
2966 ThePlantCell
Figure9. PointMutationAnalysesoftheCoreRHESequences.
(A)A1-bpinsertion(asterisk)inthelinkerregionofAtEXPA18E1greatlydecreasedthepromoteractivity.The-241deletionpromoterincludedthewild
typeor1-bpinsertion(1-I)AtEXPA18E1.
(B)A1-bpdeletion(asterisk)inthelinkerregionofAtEXPA7E1substantiallydecreasedthepromoteractivity.The-134deletionpromoterincludedthe
wildtypeor1-bpdeletion(1-D)AtEXPA7E1.ErrorbarsindicateSEin(A)and(B).
(C)TwodifferentRHEcontextsaredistinguishedbythenucleotidespacingbetweentheLPandRPofaRHE.Thelinkerspacinglengthisindicatedat
theleftoftheRHEnames.LPismoreconservedandmorepalindromictoRPintwo-nucleotidespacerRHEsthaninthree-nucleotidespacerRHEs.
PalindromicnucleotidepositionsareinthesamecolorsbetweentheLPandRP.
animals,particularlyinelementscharacterizedinanevolutionary two-andthree-spacerRHEsubgroups,thepalindromeappears
context(Bermanetal.,2004;Kellogg,2004). tobebetterestablishedinthetwo-spacerRHEsubgroup.More
In this study, we experimentally dissected root hair–specific thanfouroutofsixpositionsforasinglerepeatunitarepalin-
promoters from diverse angiosperm species, characterized a dromic in 60% of two-spacer RHEs, while fewer than three
roothair–specificcis-element(RHE)inahigh-resolutioninvivo positionsarepalindromicinmostthree-spacerRHEs(Figure9C).
analysis, and demonstrated the structural and functional con- Bothtwo-andthree-spacerRHEsoccurtogetherinthesame
servationofRHEsacrossangiosperms.Inadditiontophyloge- promoter in many cases (Figure 5), and they are equally func-
netic conservation of RHEs, the ontogenetic conservation of tional for root hair specificity as shown by diverse analyses
RHEs, despite diverse upstream regulatory manners among (Figures2and4).However,asinglenucleotideinsertionintothe
differentangiospermtaxa,isintriguing. two-spacerRHEordeletionfromthethree-spacerRHEresulted
in a large reduction in promoter activity (Figures 9A and 9B).
Thesedatasuggestthattheflexibilityinlinkerspacingbetween
RHEsCanBeDividedintoTwoSyntacticSubgroups
LPandRPneedstobeunderstoodinthesyntacticcontextof
TheRHEconsensusconsistsoftwopartslinkedbyaspacer:RP each RHE subgroup. Different linker spacings between two
and LP. While the RP of all RHEs includes the hallmark motif repeatedcis-elementsmayallowtranscriptionfactorstodimer-
CACG, the LP appears less conserved. However, when RHEs izeindifferentcombinations(Latchman,2004).Subtlesyntactic
wereseparatelygroupedintotwo-andthree-nucleotidespacer differencesbetweentwoRHEsubgroupsmightbedistinguished
RHEs, different conservation patternswere observed between bystructurallyandfunctionallyrelatedmembersofatranscrip-
thetwosubgroups(Figure9C).Intwo-spacerRHEs,thedom- tionfactorfamily(hereaftercalledRHFforroothairfactor).The
inant nucleotide for positions 3 and 7 is C, and position 3, in RHEs of At EXPA7, AtEXPA18 , and At PRP3 are not only
particular, ishighlyconserved, whereas aC in thesepositions responsible for root hair specificity but also for responses to
occursveryrarelyinthree-spacerRHEs.Bycontrast,inthree- hormonal and environmental stimuli (Bernhardt and Tierney,
spacerRHEs,position4isdominatedbyG,whichoccursonlyin 2000; Cho and Cosgrove, 2002). The hormonal/environmental
E2ofGraminaceaeandbalsamEXPA7orthologsintwo-spacer stimuli ultimately induce root hair–specific expression of these
RHEs. LP and RP are in a weak inverted repeat (palindromic) genes.Thisstimuli-mediatedsignalingpathwaymightalsoreg-
relationship.BecauseofthebiasedconservationinLPbetween ulateRHF-mediatedroothairgeneexpression.
ConservationofaRootHaircis-Element 2967
RHE-ContainingRootHairGenesAreLikelytoBe andaWD40protein(TRANSPARENTTESTAGLABRA)(Grierson
DownstreamGenesMediatingHairMorphogenesis and Schiefelbein, 2002; Schiefelbein, 2003; Schiefelbein and
Lee,2006).Inearlyrootepidermaldevelopment, thesefactors
TheRHE-containingroothair–specificgenesidentifiedthusfar
areactive in both the hair(H) and non-hair(N) cells. However,
encodecellwall–relatedproteins,suchasexpansins,LRXs,and
laterindevelopment,theoutputofthefactorsintheNposition
PRPs. Althoughnotroothaircellspecific, AtCSLD3(KOJAK),
unilaterallyinhibitstheinteractionofthefactorsintheHposition,
encodingacellulosesynthase-likeprotein,isexpressedprefer-
whichreleasestheinhibitionofroothairmorphogenesisinthe
entiallyinroothaircells(Faveryetal.,2001).Theproximalregion
Hposition.Thechangesinactivityareestablishedbypositional
oftheAtCSLD3promoterincludestwoRHE-likeelements(data
cues from underlying cortical cells (Schiefelbein, 2003), where
not shown), which do not match the RHE consensus at a few
thereceptor-likekinaseSCRAMBLED(SCM)isacandidatefor
noncriticalpositionsbutcouldformfunctionalelementswhenthe
generatingormodulatingthecue(Kwaketal.,2005).Anotherline
1-bpsubstitutionresultsfromAtEXPA7E1areconsidered(Fig-
ofevidenceimplicatesposition-dependentchromatinreorgani-
ure 2E). The root hair morphogenetic process consists of hair
zationinType3patterning(CostaandShaw,2006).
initiation(orbulgeformation)andtipgrowth,whichisnecessarily
Type2fatedeterminantsmediatingasymmetriccelldivision
accompaniedbyintensivecellwallmodification,synthesis,and
needtobecharacterized.Incontrastwiththewell-characterized
assembly(GriersonandSchiefelbein,2002).Theabovecellwall
examplesinanimalsystems,themachineryforasymmetriccell
proteinsarerepresentativeofthecellwalldynamicsduringroot
division in plant cells remains largely unknown. However, re-
hairdevelopment:expansinsforwallloosening(Balusˇkaetal.,
gardlessofthemechanismunderlyingtheunequalcelldivision,
2000; Cho and Cosgrove, 2002), CSLD3 for wall synthesis
thedivisionultimatelyresultsinanasymmetricaldistributionof
(Favery et al., 2001), and LRXs (Baumberger et al., 2003) and
cellfatedeterminantsbetweenthetwodaughtercells(Scheres
PRPs (Bernhardt andTierney, 2000) for wallassembly. Acon-
and Benfey, 1999; Betschinger and Knoblich, 2004). In animal
certedregulationofthesecellwallgeneswouldberequiredto
cells,themachineryforasymmetriccelldivisionisconservedin
implement root hair development efficiently. All these RHE-
differentspeciesandcelltypes,butthefatedeterminantsarenot
containing promoters are active from hair initiation through tip
(Betschinger and Knoblich, 2004), indicating that different cell
growth(BernhardtandTierney,2000;ChoandCosgrove,2002;
typesrequireadifferentsetoffatedeterminantstoestablishthe
Baumbergeretal.,2003),suggestingthatthegenestheycontrol
asymmetry. Known cell fate determinants are diverse, ranging
areinstrumentalinroothairdevelopment.
from transcriptional regulators to protein degradation compo-
nents(BetschingerandKnoblich,2004).
RHEHasBeenFunctionallyConservedthrough
AngiospermDivergenceofRootEpidermalPatterning
Theroothairhasbeenabasiccellularstructurefor400million
yearsinthetracheophytelineage,indicatingitsimportantrolein
the successful adaptation of land plants (Peterson, 1992). Our
study identified the functional conservation of RHE in ortholo-
gousgenesfrombothGraminaceae andeudicots.Thesedata
indicatethattheoccurrenceofRHEpredatedthedivergenceof
angiosperms(170to235millionyearsago;Yangetal.,1999)and
areconsistentwiththedivergencetime(;270millionyearsago)of
theRHE-containingOsEXPA17,whichisestimatedbyadopting
therateof9310(cid:2)10nonsynonymoussubstitutions(cid:3)site(cid:2)1(cid:3)year(cid:2)1
forgrassnucleargenes(Gaut,1998;Muse,2000).Thesecollec-
tivelyimplyastrongfunctionalconstraintonRHE.BecauseType2
roothairdistributionisfoundinthemostprimitivetracheophytes,
suchasLycopodiumandSelaginella(Dolan,1996;Figure1B),it
will be interesting to know whether RHEs are also associated
withthese.
Leavitt (1904) originally distinguished two different root hair
distributiontypes;currently,threebasictypesarerecognizedin
vascularplants(Cormack,1935,1947;DolanandCosta,2001).
Atthecellularlevel,fate-determiningmechanismsforthethree
types ofpatterns arewell defined: Type1 contains nodistinct
trichoblastcellsandthusgeneratesarandomdistribution,Type
2 is generated by asymmetric cell division, and Type 3 is cell Figure 10. Model Illustrating How Root Hair Cell Specificity of Gene
positiondependent(DolanandCosta,2001). ExpressionIsDeterminedinDifferentHairPatterningTypes.
TranscriptionalregulationinType3fatedeterminationresults There may be orthologous RHFs, the RHE binding factors, that can
from the interaction between MYBs (WEREWOLF and CA- interact with different signaling pathways activated by different fate-
PRICE), basic helix-loop-helixes (GL3/ENHANCER OF GL3), determiningmachineries.